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Auto Tag: Genome assembly

September 22, 2019

Screening and genomic characterization of filamentous hemagglutinin-deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Characterization of plasmids harboring blaCTX-M and blaCMY genes in E. coli from French broilers.

Resistance to extended-spectrum cephalosporins (ESC) is a global health issue. The aim of this study was to analyze and compare plasmids coding for resistance to ESC isolated from 16 avian commensal and 17 avian pathogenic Escherichia coli (APEC) strains obtained respectively at slaughterhouse or from diseased broilers in 2010-2012. Plasmid DNA was used to transform E. coli DH5alpha, and the resistances of the transformants were determined. The sequences of the ESC-resistance plasmids prepared from transformants were obtained by Illumina (33 plasmids) or PacBio (1 plasmid). Results showed that 29 of these plasmids contained the blaCTX-M-1 gene and belonged to the IncI1/ST3 type, with 27 and 20 of them carrying the sul2 or tet(A) genes respectively. Despite their diverse origins, several plasmids showed very high percentages of identity. None of the blaCTX-M-1-containing plasmid contained APEC virulence genes, although some of them were detected in the parental strains. Three plasmids had the blaCMY-2 gene, but no other resistance gene. They belonged to IncB/O/K/Z-like or IncFIA/FIB replicon types. The blaCMY-2 IncFIA/FIB plasmid was obtained from a strain isolated from a diseased broiler and also containing a blaCTX-M-1 IncI1/ST3 plasmid. Importantly APEC virulence genes (sitA-D, iucA-D, iutA, hlyF, ompT, etsA-C, iss, iroB-E, iroN, cvaA-C and cvi) were detected on the blaCMY-2 plasmid. In conclusion, our results show the dominance and high similarity of blaCTX-M-1 IncI1/ST3 plasmids, and the worrying presence of APEC virulence genes on a blaCMY-2 plasmid.

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September 22, 2019

In situ analyses directly in diarrheal stool reveal large variations in bacterial load and active toxin expression of enterotoxigenic Escherichia coli and Vibrio cholerae.

The bacterial pathogens enterotoxigenicEscherichia coli(ETEC) andVibrio choleraeare major causes of diarrhea. ETEC causes diarrhea by production of the heat-labile toxin (LT) and heat-stable toxins (STh and STp), whileV. choleraeproduces cholera toxin (CT). In this study, we determined the occurrence and bacterial doses of the two pathogens and their respective toxin expression levels directly in liquid diarrheal stools of patients in Dhaka, Bangladesh. By quantitative culture and real-time quantitative PCR (qPCR) detection of the toxin genes, the two pathogens were found to coexist in several of the patients, at concentrations between 102and 108bacterial gene copies per ml. Even in culture-negative samples, gene copy numbers of 102to 104of either ETEC orV. choleraetoxin genes were detected by qPCR. RNA was extracted directly from stool, and gene expression levels, quantified by reverse transcriptase qPCR (RT-qPCR), of the genes encoding CT, LT, STh, and STp showed expression of toxin genes. Toxin enzyme-linked immunosorbent assay (ELISA) confirmed active toxin secretion directly in the liquid diarrhea. Analysis of ETEC isolates by multiplex PCR, dot blot analysis, and genome sequencing suggested that there are genetic ETEC profiles that are more commonly found as dominating single pathogens and others that are coinfectants with lower bacterial loads. The ETEC genomes, including assembled genomes of dominating ETEC isolates expressing LT/STh/CS5/CS6 and LT/CS7, are provided. In addition, this study highlights an emerging important ETEC strain expressing LT/STp and the novel colonization factor CS27b. These findings have implications for investigations of pathogenesis as well as for vaccine development. IMPORTANCEThe cause of diarrheal disease is usually determined by screening for several microorganisms by various methods, and sole detection is used to assign the agent as the cause of disease. However, it has become increasingly clear that many infections are caused by coinfections with several pathogens and that the dose of the infecting pathogen is important. We quantified the absolute numbers of enterotoxigenicE. coli(ETEC) andVibrio choleraedirectly in diarrheal fluid. We noted several events where both pathogens were found but also a large dose dependency. In three samples, we found ETEC as the only pathogen sought for. These isolates belonged to globally distributed ETEC clones and were the dominating species in stool with active toxin expression. This suggests that certain superior virulent ETEC lineages are able to outcompete the gut microbiota and be the sole cause of disease and hence need to be specifically monitored.

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September 22, 2019

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome.

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota.CandidatusNitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome ofCa.N. cavascurensis harbors anamoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although abona fidegene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism.Ca.N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

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September 22, 2019

The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology.

We report a draft assembly of the genome of Hi5 cells from the lepidopteran insect pest,Trichoplusia ni, assigning 90.6% of bases to one of 28 chromosomes and predicting 14,037 protein-coding genes. Chemoreception and detoxification gene families revealT. ni-specific gene expansions that may explain its widespread distribution and rapid adaptation to insecticides. Transcriptome and small RNA data from thorax, ovary, testis, and the germline-derived Hi5 cell line show distinct expression profiles for 295 microRNA- and >393 piRNA-producing loci, as well as 39 genes encoding small RNA pathway proteins. Nearly all of the W chromosome is devoted to piRNA production, andT. nisiRNAs are not 2´-O-methylated. To enable use of Hi5 cells as a model system, we have established genome editing and single-cell cloning protocols. TheT. nigenome provides insights into pest control and allows Hi5 cells to become a new tool for studying small RNAs ex vivo.© 2018, Fu et al.

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September 22, 2019

Comparative genomics of completely sequenced Lactobacillus helveticus genomes provides insights into strain-specific genes and resolves metagenomics data down to the strain level.

Although complete genome sequences hold particular value for an accurate description of core genomes, the identification of strain-specific genes, and as the optimal basis for functional genomics studies, they are still largely underrepresented in public repositories. Based on an assessment of the genome assembly complexity for all lactobacilli, we used Pacific Biosciences’ long read technology to sequence and de novo assemble the genomes of three Lactobacillus helveticus starter strains, raising the number of completely sequenced strains to 12. The first comparative genomics study for L. helveticus-to our knowledge-identified a core genome of 988 genes and sets of unique, strain-specific genes ranging from about 30 to more than 200 genes. Importantly, the comparison of MiSeq- and PacBio-based assemblies uncovered that not only accessory but also core genes can be missed in incomplete genome assemblies based on short reads. Analysis of the three genomes revealed that a large number of pseudogenes were enriched for functional Gene Ontology categories such as amino acid transmembrane transport and carbohydrate metabolism, which is in line with a reductive genome evolution in the rich natural habitat of L. helveticus. Notably, the functional Clusters of Orthologous Groups of proteins categories “cell wall/membrane biogenesis” and “defense mechanisms” were found to be enriched among the strain-specific genes. A genome mining effort uncovered examples where an experimentally observed phenotype could be linked to the underlying genotype, such as for cell envelope proteinase PrtH3 of strain FAM8627. Another possible link identified for peptidoglycan hydrolases will require further experiments. Of note, strain FAM22155 did not harbor a CRISPR/Cas system; its loss was also observed in other L. helveticus strains and lactobacillus species, thus questioning the value of the CRISPR/Cas system for diagnostic purposes. Importantly, the complete genome sequences proved to be very useful for the analysis of natural whey starter cultures with metagenomics, as a larger percentage of the sequenced reads of these complex mixtures could be unambiguously assigned down to the strain level.

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September 22, 2019

Microbiome and infectivity studies reveal complex polyspecies tree disease in Acute Oak Decline.

Decline-diseases are complex and becoming increasingly problematic to tree health globally. Acute Oak Decline (AOD) is characterized by necrotic stem lesions and galleries of the bark-boring beetle, Agrilus biguttatus, and represents a serious threat to oak. Although multiple novel bacterial species and Agrilus galleries are associated with AOD lesions, the causative agent(s) are unknown. The AOD pathosystem therefore provides an ideal model for a systems-based research approach to address our hypothesis that AOD lesions are caused by a polymicrobial complex. Here we show that three bacterial species, Brenneria goodwinii, Gibbsiella quercinecans and Rahnella victoriana, are consistently abundant in the lesion microbiome and possess virulence genes used by canonical phytopathogens that are expressed in AOD lesions. Individual and polyspecies inoculations on oak logs and trees demonstrated that B. goodwinii and G. quercinecans cause tissue necrosis and, in combination with A. biguttatus, produce the diagnostic symptoms of AOD. We have proved a polybacterial cause of AOD lesions, providing new insights into polymicrobial interactions and tree disease. This work presents a novel conceptual and methodological template for adapting Koch’s postulates to address the role of microbial communities in disease.

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September 22, 2019

Characterization of ß-glucan formation by Lactobacillus brevis TMW 1.2112 isolated from slimy spoiled beer.

Despite several hurdles, which hinder bacterial growth in beer, certain bacteria are still able to spoil beer. One type of spoilage is characterized by an increased viscosity and slimy texture caused by exopolysaccharide (EPS) formation of lactic acid bacteria (LAB). In this study, we characterize for the first time EPS production in a beer-spoiling strain (TMW 1.2112) of Lactobacillus brevis, a species commonly involved in beer spoilage. The strain’s growth dynamics were assessed and we found an increased viscosity or ropiness in liquid or on solid media, respectively. Capsular polysaccharides (CPS) and released EPS from the cells or supernatant, respectively, were analyzed via NMR spectroscopy and methylation analysis. Both are identical ß-(1?3)-glucans, which are ramified with ß-glucose residues at position O2. Therefore, we assume that this EPS is mainly produced as CPS and partially released into the surrounding medium, causing viscosity of e.g. beer. CPS formation was confirmed via an agglutination test. A plasmid-located glycosyltransferase-2 was found as responsible for excess ß-glucan formation, chromosomal glucanases were proposed for its degradation. The glycosyltransferase-2 gene could also be specifically identified in beer-spoiling, slime-producing Lactobacillus rossiae and Lactobacillus parabuchneri strains, suggesting it as promising marker gene for the early detection of ß-glucan-producing Lactobacilli in breweries. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

Redkmer: An Assembly-Free Pipeline for the Identification of Abundant and Specific X-Chromosome Target Sequences for X-Shredding by CRISPR Endonucleases.

CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae, including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.

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September 22, 2019

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a “Y. ruckeri invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

Molecular characterization of NBS-LRR genes in the soybean Rsv3 locus reveals several divergent alleles that likely confer resistance to the soybean mosaic virus.

The divergence patterns of NBS – LRR genes in soybean Rsv3 locus were deciphered and several divergent alleles ( NBS_C, NBS_D and Columbia NBS_E ) were identified as the likely functional candidates of Rsv3. The soybean Rsv3 locus, which confers resistance to the soybean mosaic virus (SMV), has been previously mapped to a region containing five nucleotide binding site-leucine-rich repeats (NBS-LRR) genes (referred to as nbs_A-E) in Williams 82. In resistant cultivars, however, the number of NBS-LRR genes in this region and their divergence from susceptible alleles remain unclear. In the present study, we constructed and screened a bacterial artificial chromosome (BAC) library for an Rsv3-possessing cultivar, Zaoshu 18. Sequencing two positive BAC inserts on the Rsv3 locus revealed that Zaoshu 18 possesses the same gene content and order as Williams 82, but two of the NBS-LRR genes, NBS_C and NBS_D, exhibit distinct features that were not observed in the Williams 82 alleles. Obtaining these NBS-LRR genes from eight additional cultivars demonstrated that the NBS_A-D genes diverged into two different alleles: the nbs_A-D alleles were associated with the rsv3-type cultivars, whereas the NBS_A-D alleles were associated with the Rsv3-possessing cultivars. For the NBS_E gene, the cultivar Columbia possesses an allele (NBS_E) that differed from that in Zaoshu 18 and rsv3-type cultivars (nbs_E). Exchanged fragments were further detected on alleles of the NBS_C-E genes, suggesting that recombination is a major force responsible for allele divergence. Also, the LRR domains of the NBS_C-E genes exhibited extremely strong signals of positive selection. Overall, the divergence patterns of the NBS-LRR genes in Rsv3 locus elucidated by this study indicate that not only NBS_C but also NBS_D and Columbia NBS_E are likely functional alleles that confer resistance to SMV.

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September 22, 2019

Genomics: Next regeneration sequencing for reference genomes.

Various species have remarkable abilities to regenerate body parts or entire organisms after injury, but a comprehensive understanding of the molecular basis of regeneration mech- anisms will require detailed genomic resources. Two new studies report high-quality reference genomes for two classic regeneration model organ- isms with contrasting genome sizes: the axolotl salamander Ambystoma mexicanum and the planarium flatworm Schmidtea mediterranea.

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September 22, 2019

A hybrid-hierarchical genome assembly strategy to sequence the invasive golden mussel Limnoperna fortunei.

For more than 25 years, the golden mussel Limnoperna fortunei has aggressively invaded South American freshwaters, having travelled more than 5,000 km upstream across five countries. Along the way, the golden mussel has outcompeted native species and economically harmed aquaculture, hydroelectric powers, and ship transit. We have sequenced the complete genome of the golden mussel to understand the molecular basis of its invasiveness and search for ways to control it.We assembled the 1.6 Gb genome into 20548 scaffolds with an N50 length of 312 Kb using a hybrid and hierarchical assembly strategy from short and long DNA reads and transcriptomes. A total of 60717 coding genes were inferred from a customized transcriptome-trained AUGUSTUS run. We also compared predicted protein sets with those of complete molluscan genomes, revealing an exacerbation of protein-binding domains in L. fortunei. Conclusions: We built one of the best bivalve genome assemblies available using a cost-effective approach using Illumina pair-end, mate pair, and PacBio long reads. We expect that the continuous and careful annotation of L. fortunei’s genome will contribute to the investigation of bivalve genetics, evolution, and invasiveness, as well as to the development of biotechnological tools for aquatic pest control.© The Authors 2017. Published by Oxford University Press.

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September 22, 2019

Topical antibiotic use coselects for the carriage of mobile genetic elements conferring resistance to unrelated antimicrobials in Staphylococcus aureus.

Topical antibiotics, such as mupirocin and fusidic acid, are commonly used in the prevention and treatment of skin infections, particularly those caused by staphylococci. However, the widespread use of these agents is associated with increased resistance to these agents, potentially limiting their efficacy. Of particular concern is the observation that resistance to topical antibiotics is often associated with multidrug resistance, suggesting that topical antibiotics may play a role in the emergence of multidrug-resistant (MDR) strains. New Zealand (NZ) has some of the highest globally recorded rates of topical antibiotic usage and resistance. Using a combination of Pacific Biosciences single-molecule real-time (SMRT) whole-genome sequencing, Illumina short-read sequencing, and Bayesian phylogenomic modeling on 118 new multilocus sequence type 1 (ST1) communityStaphylococcus aureusisolates from New Zealand and 61 publically available international ST1 genome sequences, we demonstrate a strong correlation between the clinical introduction of topical antibiotics and the emergence of MDR ST1S. aureusWe also providein vitroexperimental evidence showing that exposure to topical antibiotics can lead to the rapid selection of MDRS. aureusisolates carrying plasmids that confer resistance to multiple unrelated antibiotics, from within a mixed population of competitor strains. These findings have important implications regarding the impact of the indiscriminate use of topical antibiotics. Copyright © 2018 Carter et al.

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September 22, 2019

Cytogenomic analysis of several repetitive DNA elements in turbot (Scophthalmus maximus).

Repetitive DNA plays a fundamental role in the organization, size and evolution of eukaryotic genomes. The sequencing of the turbot revealed a small and compact genome, as in all flatfish studied to date. The assembly of repetitive regions is still incomplete because it is difficult to correctly identify their position, number and array. The combination of classical cytogenetic techniques along with high quality sequencing is essential to increase the knowledge of the structure and composition of these sequences and, thus, of the structure and function of the whole genome. In this work, the in silico analysis of H1 histone, 5S rDNA, telomeric and Rex repetitive sequences, was compared to their chromosomal mapping by fluorescent in situ hybridization (FISH), providing a more comprehensive picture of these elements in the turbot genome. FISH assays confirmed the location of H1 in LG8; 5S rDNA in LG4 and LG6; telomeric sequences at the end of all chromosomes whereas Rex elements were dispersed along most chromosomes. The discrepancies found between both approaches could be related to the sequencing methodology applied in this species and also to the resolution limitations of the FISH technique. Turbot cytogenomic analyses have proven to add new chromosomal landmarks in the karyotype of this species, representing a powerful tool to investigate targeted genomic sequences or regions in the genetic and physical maps of this species. Copyright © 2017 Elsevier B.V. All rights reserved.

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